Genome-Wide Identification of Glutathione S-Transferase Family from Dendrobium officinale and the Functional Characterization of DoGST5 in Cadmium Tolerance

Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H2O2 and O2− levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress.


Introduction
Dendrobium officinale (D. officinale), a renowned traditional Chinese medicinal herb, is widely distributed in subtropical regions [1].It is considered the most precious species of the genus Dendrobium and is known for its diverse therapeutic effects [2].In China, fresh D. officinale can be used as a high-quality dietary supplement and is processed into various products, such as D. officinale juice, functional wine, tea, capsules, and D. officinale powder, according to consumer preference.Currently, D. officinale cultivation spans over 6000 hectares nationwide, producing over 20,000 tons of fresh product annually [3,4].However, D. officinale currently suffers from cadmium (Cd) toxicity.Field survey results indicate that the comprehensive pollution index of the heavy metal, Cd, in the cultivation substrate of D. officinale is 1.33, causing significant damage to the crop [5].
GST genes are involved in various physiological and developmental processes, as well as in the responses to biotic and abiotic stresses in numerous plant species [21][22][23][24][25].The tau and phi classes have been extensively studied in the GST gene family.Overexpression of certain members of these two classes has been demonstrated to improve tolerance to drought, salt, oxidative stress, herbicides, heavy metals, and ultraviolet (UV) stressors in transgenic plants [26][27][28][29][30], indicating their protective role against abiotic stress.Increased transcript levels of tau class genes NbGSTU1 and NbGSTU3 were observed in Nicotiana benthamiana infected with Colletotrichum [31].A recent study demonstrated that members of the tau class exhibit scavenging activity against highly toxic reactive carbonyl species (RCS), such as acrolein, and suggested their role in the defense system against RCS [32].Genes from these two classes have also been implicated in the transport and metabolism of secondary compounds, such as Bz2 in maize [33], An9 in petunia [34], and TT19 in Arabidopsis [35].Plants overexpressing GST genes, such as Nt107 in tobacco and LeGSTU2 in Arabidopsis, have demonstrated increased tolerance to various stresses [36,37].
Advancements in whole-genome sequencing and genome-wide analysis have enabled the identification and characterization of the GST gene family in various plant species.Research findings have revealed that GSTs, as a multigene family, have varying genes across different species.In Arabidopsis, 53 GST family members have been identified [38].Seventy-nine members have been found in rice [12], 59 in Gossypium raimondii, 49 in G. arboreum [18], 90 in tomato [11], 54 in peach [39], 69 in apple [40], 97 in kiwifruit [41], and 57 in pear [42].Although the GST gene family has been extensively characterized in various plant species, a comprehensive analysis of the GST family in D. officinale has not been reported.This study aimed to identify GST family members in D. officinale and analyze their expression patterns in response to Cd exposure.Subsequently, genes exhibiting significant responsiveness were selected for functional validation, laying the groundwork for further exploration of the mechanisms underlying Cd resistance mediated by DoGST family genes.

Identification of the GST Protein Family in D. officinale
A total of 138 known GST protein sequences from Arabidopsis and rice (56 and 82 sequences, respectively) were obtained to construct a Hidden Markov Model (HMM) for a comprehensive understanding of the sequence characteristics of the GST family.Subsequently, the obtained sequences were processed using the Pfam A database and PfamScan software (version 1.6) for annotation, with the requirement that the selected sequences contain only two specific structures: GST_N and GST_C.Consequently, 46 members of the GST family were identified in D. officinale (Table S1).

Phylogenetic Analysis of the GST Gene Family
Phylogenetic analysis of the GST gene family protein sequences from D. officinale, rice, and Arabidopsis was performed using the maximum likelihood method in MEGA software.After comparing the results, the GST proteins were categorized into nine groups, including phi, EF1Bγ, theta, TCHQD, zeta, GHR, lambda, DHAR, and tau.Among them, the tau branch comprised 22 family members, accounting for 47.8% and representing the largest branch of the GST gene family in D. officinale.The phi, EF1Bγ, theta, TCHQD, zeta, GHR, lambda, and DHAR branches contained 11, 2, 2, 1, 3, 0, 3, and 2 family members, respectively (Figure 1).

Physicochemical Property Analysis of the GST Gene Family
The ExPASy online platform was used to predict the physicochemical propertie the 46 members of the GST gene family in D. officinale to obtain data on molecular we and other aspects (Table S2).The amino acid length ranged from 124 to 506, while molecular weight ranged from 14,034.32 to 57,214.76 kilodaltons, and the isoelectric p ranged  S2).

Analysis of Gene Structure and Conserved Protein Motifs in the GST Gene Family
Exons and introns play vital roles in understanding evolutionary relationsh among species based on their positional information.The figure above depicts a com hensive analysis of all of the identified genes.Genes generally contained 1-10 exons, w genes 23 and 25 having the largest number of exons, each containing 10 exons.Convers

Physicochemical Property Analysis of the GST Gene Family
The ExPASy online platform was used to predict the physicochemical properties of the 46 members of the GST gene family in D. officinale to obtain data on molecular weight and other aspects (Table S2).The amino acid length ranged from 124 to 506, while the molecular weight ranged from 14,034.32 to 57,214.76 kilodaltons, and the isoelectric point ranged from 4.94 to 9.40.Eleven proteins, including DoGST2, DoGST27, DoGST35, DoGST45, DoGST19, DoGST26, DoGST24, DoGST37, DoGST3, DoGST12, and DoGST7, exhibited isoelectric points > 7, indicating alkaline proteins.However, the rest were acidic proteins with isoelectric points < 7. The instability index ranged  S2).

Analysis of Gene Structure and Conserved Protein Motifs in the GST Gene Family
Exons and introns play vital roles in understanding evolutionary relationships among species based on their positional information.The figure above depicts a comprehensive analysis of all of the identified genes.Genes generally contained 1-10 exons, with genes 23 and 25 having the largest number of exons, each containing 10 exons.Conversely, DoGST22 and DoGST36 had the lowest number of exons, each containing only one exon (Figure 2A).The MEME online tool for domain analysis results indicated that the GST family in D. officinale possessed 15 types of motifs.Each member contained at least two GST domains.DoGST22 and DoGST36 had the lowest number of exons, each containing only one e (Figure 2A).The MEME online tool for domain analysis results indicated that the G family in D. officinale possessed 15 types of motifs.Each member contained at least GST domains.Specifically, DoGST37 had the fewest motifs, containing only 2 mo while DoGST15 had the most, containing 13 motifs (Figure 2B).

Subcellular Localization Prediction of GST Proteins
Subcellular localization prediction was performed for the 46 members of the G gene family in D. officinale using online tools.The results revealed that DoGST7 was dicted to localize to the plasma membrane.DoGST2, DoGST21, DoGST23, DoGST24, DoGST25 were predicted to localize to the chloroplast.DoGST4, DoGST9, DoGST26,

Subcellular Localization Prediction of GST Proteins
Subcellular localization prediction was performed for the 46 members of the GST gene family in D. officinale using online tools.The results revealed that DoGST7 was predicted to localize to the plasma membrane.DoGST2, DoGST21, DoGST23, DoGST24, and DoGST25 were predicted to localize to the chloroplast.DoGST4, DoGST9, DoGST26, and DoGST37 were predicted to localize to the extracellular space, indicating secretion proteins.DoGST1, DoGST3, DoGST8, DoGST14, DoGST15, DoGST28, DoGST29, DoGST30, DoGST39, DoGST41, DoGST42, DoGST43, and DoGST45 were predicted to localize to the nucleus.The remaining 23 proteins were predicted to localize to the cytoplasm (Table S3).

Prediction of TF Binding Sites on the Promoters of the GST Gene Family
The prediction results for the promoter elements revealed that the 46 members of the GST gene family in D. officinale contained a rich variety of cis-acting elements (Figure 3).These primarily included abiotic stress-related, hormone-responsive, and light-responsive elements.Notably, the promoters of DoGST13 and DoGST6 contained the highest number of cis-acting elements.These elements provided references for the future selection of upstream regulatory factor promoter fragments and interaction sites between regulatory factors and promoters (Figure 3).DoGST37 were predicted to localize to the extracellular space, indicating secretion proteins.DoGST1, DoGST3, DoGST8, DoGST14, DoGST15, DoGST28, DoGST29, DoGST30, DoGST39, DoGST41, DoGST42, DoGST43, and DoGST45 were predicted to localize to the nucleus.The remaining 23 proteins were predicted to localize to the cytoplasm (Table S3).

Prediction of TF Binding Sites on the Promoters of the GST Gene Family
The prediction results for the promoter elements revealed that the 46 members of the GST gene family in D. officinale contained a rich variety of cis-acting elements (Figure 3).These primarily included abiotic stress-related, hormone-responsive, and light-responsive elements.Notably, the promoters of DoGST13 and DoGST6 contained the highest number of cis-acting elements.These elements provided references for the future selection of upstream regulatory factor promoter fragments and interaction sites between regulatory factors and promoters (Figure 3).

Analysis of Expression Patterns of the GST Gene Family in Response to Cd
The relative expression levels of GST genes in response to Cd stress were analyzed using transcriptome data from our previous research [43].The results revealed that 23 GST family genes were generally downregulated in response to Cd, while the remaining 23 were upregulated (Figure 4).Furthermore, we found that the expression levels of DoGST5, DoGST11, and DoGST33 increased with increasing Cd treatment time, while the expression levels of DoGST15, DoGST18, DoGST19, DoGST23, DoGST28, DoGST30, DoGST41, and DoGST43 initially increased and then decreased after Cd treatment (Figure 4).This suggested that these genes may be involved in the Cd tolerance response of D. officinale and may be candidate genes for further analysis of Cd tolerance mechanisms.

Analysis of Expression Patterns of the GST Gene Family in Response to Cd
The relative expression levels of GST genes in response to Cd stress were analyzed using transcriptome data from our previous research [43].The results revealed that 23 GST family genes were generally downregulated in response to Cd, while the remaining 23 were upregulated (Figure 4).Furthermore, we found that the expression levels of DoGST5, DoGST11, and DoGST33 increased with increasing Cd treatment time, while the expression levels of DoGST15, DoGST18, DoGST19, DoGST23, DoGST28, DoGST30, DoGST41, and DoGST43 initially increased and then decreased after Cd treatment (Figure 4).This suggested that these genes may be involved in the Cd tolerance response of D. officinale and may be candidate genes for further analysis of Cd tolerance mechanisms.

Subcellular Localization Analysis of DoGST5 Protein
Based on the expression pattern analysis, we selected the D. officinale GST family gene DoGST5, which showed a significant response to Cd, for further analysis.Firstly, the subcellular localization of this gene was analyzed.In this study, a fusion protein expression vector of DoGST5 protein fused with GFP protein (p35S::DoGST5::GFP) was constructed.Then, the recombinant plasmid was transferred into tobacco epidermal cells via Agrobacterium-mediated transformation and observed using confocal laser scanning microscopy.The results indicated that DoGST5 protein was expressed in the cytoplasm of tobacco epidermal cells, indicating that DoGST5 was localized in the cytoplasm (Figure 5).This was consistent with the predicted subcellular localization of DoGST5 protein.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 14 Based on the expression pattern analysis, we selected the D. officinale GST family gene DoGST5, which showed a significant response to Cd, for further analysis.Firstly, the subcellular localization of this gene was analyzed.In this study, a fusion protein expression vector of DoGST5 protein fused with GFP protein (p35S::DoGST5::GFP) was constructed.Then, the recombinant plasmid was transferred into tobacco epidermal cells via Agrobacterium-mediated transformation and observed using confocal laser scanning microscopy.The results indicated that DoGST5 protein was expressed in the cytoplasm of tobacco epidermal cells, indicating that DoGST5 was localized in the cytoplasm (Figure 5).This was consistent with the predicted subcellular localization of DoGST5 protein.

Functional Analysis of DoGST5 in Cd Tolerance
Two transgenic Arabidopsis lines overexpressing DoGST5 were identified using semi-quantitative PCR analysis.The results indicated that, at nearly identical brightness of the AtActin band, Col-0 did not exhibit amplification bands, while the two overexpression lines displayed brighter bands (Figure S1), indicating successful expression of DoGST5 in both transgenic lines, making them suitable for subsequent experiments.Seeds of wild-type and transgenic homozygous Arabidopsis lines were sown on 1/2 MS solid culture medium containing 0 and 60 µM CdCl2 and placed in a growth chamber for two weeks.Phenotypic differences were observed, photographs were taken, and the root length and biomass were measured.As shown in Figure 6A,B, on culture medium containing 60 µM CdCl2, the Arabidopsis lines overexpressing DoGST5 exhibited stronger resistance than the wild type.The root length of the overexpression lines was significantly longer than that of the wild type.The fresh weight of the overexpression lines was also significantly greater than that of the wild type.However, there were no significant differences in root length and biomass under control conditions (Figure 6C,D).

Functional Analysis of DoGST5 in Cd Tolerance
Two transgenic Arabidopsis lines overexpressing DoGST5 were identified using semiquantitative PCR analysis.The results indicated that, at nearly identical brightness of the AtActin band, Col-0 did not exhibit amplification bands, while the two overexpression lines displayed brighter bands (Figure S1), indicating successful expression of DoGST5 in both transgenic lines, making them suitable for subsequent experiments.Seeds of wild-type and transgenic homozygous Arabidopsis lines were sown on 1/2 MS solid culture medium containing 0 and 60 µM CdCl 2 and placed in a growth chamber for two weeks.Phenotypic differences were observed, photographs were taken, and the root length and biomass were measured.As shown in Figure 6A,B, on culture medium containing 60 µM CdCl 2 , the Arabidopsis lines overexpressing DoGST5 exhibited stronger resistance than the wild type.The root length of the overexpression lines was significantly longer than that of the wild type.The fresh weight of the overexpression lines was also significantly greater than that of the wild type.However, there were no significant differences in root length and biomass under control conditions (Figure 6C,D).

Enhancement of Plant Cd Tolerance by DoGST5 via Balancing ROS Levels
During GST reduction, GSH acts as an electron donor, reducing the organic peroxides in nucleotides and fatty acids to yield monohydric compounds.This reaction reduces the proportion of peroxides, effectively protects the cells, and prevents oxidative damage.Therefore, we measured the ROS content in wild-type and DoGST5 overexpression lines of Arabidopsis.The results indicated that the H2O2 and O2 − levels in both Arabidopsis lines were increased significantly compared to the control group after Cd treatment.However,

Enhancement of Plant Cd Tolerance by DoGST5 via Balancing ROS Levels
During GST reduction, GSH acts as an electron donor, reducing the organic peroxides in nucleotides and fatty acids to yield monohydric compounds.This reaction reduces the proportion of peroxides, effectively protects the cells, and prevents oxidative damage.Therefore, we measured the ROS content in wild-type and DoGST5 overexpression lines of Arabidopsis.The results indicated that the H 2 O 2 and O 2 − levels in both Arabidopsis lines were increased significantly compared to the control group after Cd treatment.However, the DoGST5 overexpression lines exhibited significantly lower H 2 O 2 and O 2 − levels than wild-type Arabidopsis after Cd treatment (Figure 6E,F), indicating that overexpression of DoGST5 could reduce the excessive accumulation of ROS induced by Cd stress in plants, thereby reducing cell damage and enhancing plant Cd tolerance.

Discussion
Plant GSTs are multifunctional small soluble proteins widely distributed in organisms.Plant GSTs catalyze the reaction of the tripeptide, glutathione, transferring it to a common substrate, thus producing polar S-glutathione reaction products.Subsequently, these metabolites are transported to vacuoles, exerting detoxification effects.In this study, 46 members of the D. officinale GST gene family were identified based on the published genome and clustered into eight major classes, including phi, EF1Bγ, theta, TCHQD, zeta, lambda, DHAR, and tau.The number of GST genes in D. officinale was lower than that in Arabidopsis (66 GST genes) [44], rice (59 GST genes) [12], poplar (81 GST genes) [15], tomato (90 GST genes) [11], and soybean (101 GST genes) [45], but higher than that in potato (42 GST genes) [10].The GST family members in Arabidopsis were divided into eight subfamilies, including phi, tau, theta, zeta, lambda, EF1Bγ, DHAR, and TCHQD [17].D. officinale contains many GST subfamilies, suggesting that the D. officinale GST family may have undergone more sequence and functional differentiation during evolution.Phylogenetic studies have demonstrated that plant GSTs evolve gradually after plant differentiation, with the tau and phi subfamilies being particularly special and widely distributed in plants [46].Tau and phi class genes play important roles in responses to salt, oxidative stress, herbicides, heavy metals, and UV stressors in plants [26][27][28][29][30].Moreover, we found that D. officinale contains 12 and 14 genes for the tau and phi classes, respectively, belonging to the largest branches, indicating their crucial roles in stress response.
A study of cis-acting elements in the GST gene promoter regions revealed that many family members include MYC elements.External abiotic stress response regulation methods primarily include two types: ABA-dependent and non-dependent types.The roles of DRE and ABRE elements are significant in these two regulatory mechanisms.The first type primarily includes MYC and MYB elements, and the signaling system uses ABA to regulate MYC and other transcription factors, thereby expressing downstream stressresistance genes [47].For the second type, when the signal activates the sensor protein in the regulation process, it is transferred to DREB, which then binds to DRE to achieve expression [48].ABRE is also important for plant abiotic stress.The induction of many specific proteins through ABA response mechanisms plays a regulatory role in plants.Consequently, these cis-acting elements may play important roles in the abiotic stress response of D. officinale.
GSTs are involved in multiple biochemical reactions and play a role in plant stress tolerance.Plant GSTs regulate reversible S-glutathionylation of protein thiol residues, protecting proteins from oxidation under stress [49].Transgenic overexpression of GSTs has provided insight into their role in abiotic stress acclimatization.Overexpression of tau class GSTs from species like Glycine soja, Arabidopsis, and Prosopis juliflora resulted in enhanced stress tolerance through oxidative stress tolerance, induction of antioxidant machinery, and changes in redox state [36,[50][51][52].Overexpression of rice OsGSTU4 led to pleiotropic effects, including reduced sensitivity to ABA and auxin, and upregulation of defense-related pathways [29].Zeta class GSTs have also contributed to abiotic stress tolerance despite lacking significant GST/GPOX activity.Overexpression of OsGSTZ2 in rice and ThGSTZ1 in Tamarix hispida increased stress tolerance and GST/GPOX activity [53][54][55][56].GSTZ isomerase activity may reduce oxidant accumulation, aiding antioxidant machinery [57].
However, there are no reports on the resistance of the GST gene family in D. officinale.This study is the first time to report expression analysis of the GST gene family in D. officinale under Cd stress.Differentially expressed genes can serve as genetic resources for breeding D. officinale with heavy metal tolerance.The Cd-induced significant up-regulation of the DoGST5 gene was preliminarily validated for Cd tolerance.Our evolutionary analysis showed that DoGST5 belongs to the tau family.Overexpression of DoGST5 in Arabidopsis increased its tolerance to Cd by reducing levels of Cd-induced H 2 O 2 and O 2 − .The results of the subcellular localization analysis showed that DoGST5 was localized in the cytoplasm.Its presence in the cytoplasm could be associated with detoxifying ROS and other harmful compounds, which is crucial for maintaining cellular homeostasis.The results indicated that overexpression of DoGST5 can reduce excessive accumulation of ROS induced by Cd stress in plants, thereby alleviating plant cell damage and enhancing plant Cd tolerance.The studies mentioned above suggested that the protective functions of GSTs against abiotic stress are linked to protein abundance and catalytic activity.However, silencing AtGSTU17 resulted in increased drought and salt stress tolerance, accompanied by anatomical and physiological changes such as a smaller stomatal aperture, lower transpiration rate, increased root growth, and higher ABA and GSH contents [58].These findings highlight the signaling functions of GSTs beyond their catalytic activities, offering potential for enhancing plant robustness under abiotic stress.

Identification of Family Member Proteins
Based on the GST protein family sequences, a Hidden Markov Model (HMM) was constructed using HMMER software (version 3.0) to search for and identify the complete coding protein sequences in D. officinale and to comprehensively understand the family.For the GST family reference sequences, a BLASTp search was conducted against all protein sequences, with an e-value threshold set to 1 × 10 −10 .Sequences that successfully matched were considered candidate sequences, combined to obtain the candidate GST family protein sequences.The obtained sequences were subjected to structural domain annotation using PfamScan software (version 1.6) and the Pfam A database to confirm sequences containing only the GST_N and GST_C domains as the final GST sequences.

Evolutionary Tree Analysis of GSTs
After identification, a series of GST protein family sequences from other plants was obtained, enabling the construction of a maximum likelihood tree.The construction method involved multiple sequence alignment using MAFFT and subsequent tree building using MEGA software.To assess the confidence of the calculated evolutionary tree branches, the parameters were set as follows: substitution model-the Jones-Taylor-Thornton (JTT) model, site coverage cutoff-95%, bootstrap-1000 replicates.The other parameters were set to default.

Motif Structure Analysis of GST Family Proteins
MEME software (http://meme.nbcr.net/meme,version 4.11.2, accessed on 20 April 2020) was employed to analyze the conserved motifs in the GST family.The parameter for motif discovery was set as 15.

Gene Structure Analysis of GST Family
The obtained GST protein sequences were submitted to the MEME online tool (https: //meme-suite.org/meme/tools/meme,accessed on 20 April 2020) to identify conserved motifs (Motifs), with the maximum value for motif parameters set to 10.The identified motifs from the protein sequences and structural information such as exons, introns, and untranslated regions (UTRs) extracted from the genome annotation files were visualized using the "Visualize Gene Structure" tool in TBtools software (version 0.6, accessed on 20 April 2020).

Subcellular Localization
The CDS of DoGST5 without a stop codon was cloned into the binary vector 35S::GFP (modified from pCAMBIA1300).The primers used are listed in Table S3.The resultant DoGST5-GFP vector was transformed into Agrobacterium tumefaciens and cultured in 4 mL of LB medium containing kanamycin resistance.The culture was shaken at 28 • C and 200 rpm until the OD 600 reached approximately 0.6.After centrifugation at 8000 rpm for 6 min, the supernatant was discarded, and the same volume of MES solution (10 mM MES with pH = 5.6, 10 mM MgCl 2 , and 0.2 mM acetosyringone) was used for resuspension.After centrifugation, the pellet was resuspended in an equal volume of MES solution and incubated at room temperature for 2 h.Tabacco (cultivated for 45 days) was injected into the backs of the leaves using a sterile syringe and incubated for three days.The GFP fluorescence of the leaves near the injection site was observed using a laser confocal microscope (LSM880: Karl Zeiss, Leica, Germany).

Arabidopsis Transgenic Experiment
The CDS of DoGST5 with a stop codon was cloned into the binary vector 35S::GFP.The primers used are listed in Table S3.The Agrobacterium carrying the 35S::DoGST5 plasmid was cultured in LB liquid medium (containing 50 µg•mL −1 kanamycin and 50 µg•mL −1 rifampicin) until the OD 600 reached approximately 0.8.The cells were centrifuged and resuspended in an inoculation solution (1/2 MS liquid medium, 5% sucrose, 0.03% Silwet L-77 (Solarbio Science & Technology, Beijing, China), 0.01 µg•mL −1 6-BA, and 20 mg•L −1 AS, pH adjusted to 5.7 with KOH).The plants were then immersed in the suspension solution for 5 min.After 24 h of dark treatment, the plants were transferred to normal growth conditions.Positive seedlings were screened on 1/2 MS solid medium containing 50 µg•mL −1 hygromycin.T3 generation seeds were obtained for subsequent experiments.The H 2 O 2 and O 2 − concentrations were measured using assay kits (Suzhou Keming Biotech Co. Ltd., Jiangsu, China).For the H 2 O 2 assay, 0.1 g of leaves was added to 1 mL of reagent 1, homogenized in an ice bath, then transferred to an EP tube, adjusted to 1 mL with reagent 1, and centrifuged at 8000× g for 10 min at 4 • C. For the O 2 − assay, 100 mg of frozen leaves was added to 1 mL of extracting solution, homogenized in an ice bath, and centrifuged at 10,000× g for 20 min at 4 • C. The absorbance of the supernatant was measured at 415 nm for H 2 O 2 and 530 nm for O 2 − .

Conclusions
In conclusion, this study investigated the GST gene family in D. officinale, particularly its response to Cd stress.We identified 46 GST genes and classified them into nine groups, with the tau group being the most prominent.Phylogenetic and physicochemical analyses revealed diverse properties of these genes.Subcellular localization predictions indicated that half of the GSTs were cytoplasmic.Expression analysis under Cd stress identified DoGST5 as significantly responsive.The transient expression of DoGST5-GFP in tobacco confirmed its cytoplasmic localization.Overexpression of DoGST5 in Arabidopsis enhanced Cd tolerance by reducing ROS levels and mitigating cell damage.Functional analyses revealed that DoGST5 overexpression improved root length and biomass under Cd stress, while ROS measurements exposed lower H 2 O 2 and O 2 − levels in transgenic lines than in wild-type plants.These findings suggest that DoGST5 plays a vital role in enhancing Cd tolerance by balancing ROS levels, offering potential applications for improving plant resilience to heavy metal stress.
from 20.01 to 55.94, with 20 proteins having indices > 40, indicating instability, and the remaining proteins having indices < 40, suggesting stability.The aliphatic index ranged from 57.06 to 111.71.The average hydrophilicity of the protein coefficient ranged from −1.216 to 0.156.DoGST9 and DoGST36 had coefficients of −1.216 and −0.563, respectively, indicating hydrophobic proteins.The remaining 44 proteins had coefficients > −0.5, indicating hydrophilic proteins (Table

Figure 2 .
Figure 2. Gene structures and conserved motifs of DoGSTs.(A) Exon-intron organization of Do genes.Exons are shown as red rectangles; introns are shown as gray lines.(B) Composition distributions of conserved motifs in DoGST proteins.The conserved motifs are represented by ferent numbers and rectangle colors.

Figure 2 .
Figure 2. Gene structures and conserved motifs of DoGSTs.(A) Exon-intron organization of DoGST genes.Exons are shown as red rectangles; introns are shown as gray lines.(B) Composition and distributions of conserved motifs in DoGST proteins.The conserved motifs are represented by different numbers and rectangle colors.

Figure 3 .
Figure 3. Prediction of TF binding sites on the promoter of GST family genes in Dendrobium officinale.The 2 kb upstream of the translation site was obtained for analysis, and the different colors represent the different of cis-acting elements in the promoter region of each DoGST gene.

Figure 3 .
Figure 3. Prediction of TF binding sites on the promoter of GST family genes in Dendrobium officinale.The 2 kb upstream of the translation site was obtained for analysis, and the different colors represent the different of cis-acting elements in the promoter region of each DoGST gene.

Figure 4 .
Figure 4. Expression analysis of GST family genes in response to Cd stress in Dendrobium officinale.T01 represents 0 mg•L −1 Cd treatment as the control.T10, T11, and T12 represent three biological repeats of 14 mg•L −1 Cd treatment for 15 days.T13, T14, and T15 represent three biological repeats of 14 mg•L −1 Cd treatment for 30 days.

Figure 4 .
Figure 4. Expression analysis of GST family genes in response to Cd stress in Dendrobium officinale.T01 represents 0 mg•L −1 Cd treatment as the control.T10, T11, and T12 represent three biological repeats of 14 mg•L −1 Cd treatment for 15 days.T13, T14, and T15 represent three biological repeats of 14 mg•L −1 Cd treatment for 30 days.

Figure 6 .
Figure 6.Overexpression of DoGST5 enhances Cd tolerance in Arabidopsis by reducing the excessive accumulation of ROS induced by Cd stress.(A,B) Phenotypic analysis of Cd tolerance in Arabidopsis overexpressing DoGST5.Col-0 and DoGST5 overexpression Arabidopsis lines were cultured on 0 and 60 µM 1/2 MS solid medium for 14 days.(C,D) Primary root length and fresh weight of Col-0 and DoGST5 overexpression lines.Data are means ± SD (n = 5).(E,F) Overexpression of DoGST5 in Arabidopsis reduces excessive ROS levels induced by Cd stress.Columns with different letters indicate significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons.

Figure 6 .
Figure 6.Overexpression of DoGST5 enhances Cd tolerance in Arabidopsis by reducing the excessive accumulation of ROS induced by Cd stress.(A,B) Phenotypic analysis of Cd tolerance in Arabidopsis overexpressing DoGST5.Col-0 and DoGST5 overexpression Arabidopsis lines were cultured on 0 and 60 µM 1/2 MS solid medium for 14 days.(C,D) Primary root length and fresh weight of Col-0 and DoGST5 overexpression lines.Data are means ± SD (n = 5).(E,F) Overexpression of DoGST5 in Arabidopsis reduces excessive ROS levels induced by Cd stress.Columns with different letters indicate significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons.